The present invention is concerned with the isolation and use of super-infective, tumor-specific, attenuated strains of parasites including, but not limited to, bacteria, fungi and protists. In certain embodiments the parasites include the bacterium Salmonella spp., such as Salmonella typhimurium, the bacterium Mycobacterium avium, and the protozoan Leishmania amazonensis, for the diagnosis and treatment of sarcomas, carcinomas, and other solid tumor cancers. In other embodiments, the present invention is concerned with the isolation and use of super-infective, tumor-specific, suicide gene-containing strains of parasites.
Citation or identification of any reference in Section 2 of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
A major problem in the chemotherapy of solid tumor cancers is the delivery of therapeutic agents, such as drugs, in sufficient concentrations to eradicate tumor cells while at the same time minimizing damage to normal cells. Thus, studies in many laboratories are directed toward the design of biological delivery systems, such as antibodies, cytokines, and viruses for targeted delivery of drugs, pro-drug converting enzymes, and/or genes into tumor cells. Houghton and Colt, 1993, New Perspectives in Cancer Diagnosis and Management 1: 65-70; de Palazzo,et al., 1992a, Cell. Immunol. 142:338-347; de Palazzo et al., 1992b, Cancer Res. 52: 5713-5719; Weiner, et al., 1993a, J. Immunotherapy 13:110-116; Weiner et al., 1993b, J. Immunol. 151:2877-2886; Adams et al., 1993, Cancer Res. 53:4026-4034; Fanger et al., 1990, FASEB J. 4:2846-2849; Fanger et al., 1991, Immunol. Today 12:51-54; Segal, et al., 1991, Ann N.Y. Acad. Sci. 636:288-294; Segal et al., 1992, Immunobiology 185:390-402; Wunderlich et al., 1992; Intl. J. Clin. Lab. Res. 22:17-20; George et al., 1994, J. Immunol. 152:1802-1811; Huston et al., 1993, Intl. Rev. Immunol. 10:195-217; Stafford et al., 1993, Cancer Res. 53:4026-4034; Haber et al., 1992, Ann. N.Y. Acad. Sci. 667:365-381; Haber, 1992, Ann. N.Y. Acad. Sci. 667: 365-381; Feloner and Rhodes, 1991, Nature 349:351-352; Sarver and Rossi, 1993, AIDS Research and Human Retroviruses 9:483-487; Levine and Friedmann, 1993, Am. J. Dis. Child 147:1167-1176; Friedmann, 1993, Mol. Genetic Med. 3:1-32; Gilboa and Smith, 1994, Trends in Genetics 10:139-144; Saito et al., 1994, Cancer Res. 54:3516-3520; Li et al., 1994, Blood 83:3403-3408; Vieweg et al., 1994, Cancer Res. 54:1760-1765; Lin et al., 1994, Science 265:666-669; Lu et al., 1994, Human Gene Therapy 5:203-208; Gansbacher et al., 1992, Blood 80:2817-2825; Gastl et al., 1992, Cancer Res. 52:6229-6236.
Because of their biospecificity, such systems could in theory deliver therapeutic agents to tumors. However, it has become apparent that numerous barriers exist in the delivery of therapeutic agents to solid tumors that may compromise the effectiveness of antibodies, cytokines, and viruses as delivery systems. Jain, 1994, Scientific American 7:58-65 (Jain). For example, in order for chemotherapeutic agents to eradicate metastatic tumor cells, they must
a) travel to the tumors via the vasculature;
b) extravasate from the small blood vessels supplying the tumor;
c) traverse through the tumor matrix to reach those tumor cells distal to the blood supply; and
d) interact effectively with the target tumor cells (adherence, invasion, pro-drug activation, etc).
Although antibodies and viruses can express specific recognition sites for tumor cells, they are dependent solely upon the forces of diffusion and convection in order to reach these sites. According to Jain:
An agent that destroys cancers cells in a culture dish should, in theory, be able to kill such cells in the body. . . . Sadly, however, the existing pharmacopoeia has not markedly reduced the number of deaths caused by the most common solid tumors in adults, among them cancers of the lung, breast, colon, rectum, prostate, and brain. . . . Before a blood-borne drug can begin to attack malignant cells in a tumor, it must accomplish three critical tasks. It has to make its way into a microscopic blood vessel lying near malignant cells in the tumor, exit from the vessel into the surrounding matrix (the interstitium), and finally, migrate through the matrix to the cells. Unfortunately, tumors often develop in ways that hinder each of these steps.
Jain points out that blood vessels supplying tumors are irregular and convoluted in shape so that blood flow is frequently restricted compared to that in normally vascularized tissue. In addition, there is an unusually high interstitial pressure in many tumors that counteracts the blood flow. Jain further points out that the two chief forces governing the transport of agents to tumor cells via the circulatory system are convection (the transport of molecules by a stream of flowing fluid), and diffusion (the movement of molecules from an area of high concentration to an area of low concentration). Since tumors are often non-uniformly vascularized, many cells in the tumors receive nutrients through the process of diffusion through the matrix. Jain and coworkers obtained data suggesting that xe2x80x9ca continuously supplied monoclonal antibody having a molecular weight of 150,000 daltons could take several months to reach a uniform concentration in a tumor that measured one centimeter in radius and had no blood supply in its center.xe2x80x9d
Regarding bacteria and cancer, an historical review reveals a number of clinical observations in which cancers were reported to regress in patients with bacterial infections. Nauts et al., 1953, Acta Medica. Scandinavica 145:1-102, (Suppl. 276) state:
The treatment of cancer by injections of bacterial products is based on the fact that for over two hundred years neoplasms have been observed to regress following acute infections, principally streptococcal. If these cases were not too far advanced and the infections were of sufficient severity or duration, the tumors completely disappeared and the patients remained free from recurrence.
Shear, 1950, J. A.M.A. 142:383-390 (Shear), observed that 75% of the spontaneous remissions in untreated leukemia in the Children""s Hospital in Boston occurred following an acute episode of bacterial infection. Shear stated:
Are pathogenic and non-pathogenic organisms one of Nature""s controls of microscopic foci of malignant disease, and in making progress in the control of infectious diseases, are we removing one of Nature""s controls of cancer?
Subsequent evidence from a number of research laboratories indicated that at least some of the anti-cancer effects are mediated through stimulation of the host immune system, resulting in enhanced immuno-rejection of the cancer cells. For example, release of the lipopolysaccharide (LPS) endotoxin by Gram negative bacteria such as Salmonella triggers release of tumor necrosis factor, TNF, by cells of the host immune system, such as macrophages, Christ et al., 1995, Science 268:80-83. Elevated TNF levels in turn initiate a cascade of cytokine-mediated reactions which culminate in the death of tumor cells. In this regard, Carswell et al., 1975, Proc. Natl. Acad. Sci. USA 72:3666-3669, demonstrated that mice injected with bacillus Calmette-Guerin (BCG) have increased serum levels of TNF and that TNF-positive serum caused necrosis of the sarcoma Meth A and other transplanted tumors in mice. Further, Klimpel et al., 1990, J. Immunol. 145:711-717, showed that fibroblasts infected in vitro with Shigella or Salmonella had increased susceptibility to TNF.
As a result of such observations as described above, immunization of cancer patients with BCG injections is currently utilized in some cancer therapy protocols. See Sosnowski, 1994, Compr. Ther. 20:695-701; Barth and Morton, 1995, Cancer 75 (Suppl. 2) :726-734; Friberg, 1993, Med. Oncol. Tumor. Pharmacother. 10:31-36 for reviews of BCG therapy.
Although the natural biospecificity and evolutionary adaptability of parasites has been recognized for some time and the use of their specialized systems as models for new therapeutic procedures has been suggested, there are few reports of, or proposals for, the actual use of parasites as vectors.
In this regard, Pidherney et al., 1993, Cancer Letters 72:91-98 (Pidherney et al.) and Alizadeh et al., 1994, Infect. Immun. 62:1298-1303 (Alizadeh et al.) have provided evidence that the pathogenic free-living amoeba, Acanthamoeba castellani, has tumorcidal capabilities toward human tumor cells, including melanoma, when added to tumor cells growing in culture or when injected directly into tumors in nude mice. Pidherney et al. conclude:
The feasibility of utilizing the tumorcidal properties of pathogenic/free-living amoebae and their cell-free products in the treatment of drug-resistant or radio-resistant tumors warrants further investigation.
However, Pidherney et al. also point out that such pathogenic/free living amoebae can exist either as free-living organisms feeding on bacteria or as opportunistic pathogens producing life-threatening meningoencephalitis or blinding keratitis.
Thus, it is readily apparent that for any parasite to be effective as a therapeutic vector, for example, for human tumors, the benefit of the parasite as a vector must outweigh its risk as a pathogen to the patient. Therefore, although Pidherney et al. and Alizadeh et al. demonstrated cytotoxicity of pathogenic amoebae toward tumor cells, and further suggested their use in the treatment of drug-resistant and radio-resistant tumors, they offered no solution for the inherent pathogenicity of these organisms once injected into cancer patients. Furthermore, they offered no method, e.g., genetic selection for isolating super-infective, tumor-specific strains of pathogenic amoebae nor did they suggest insertion into the amoebael genome of genetic constructs containing inducible genes for the synthesis and secretion of pro-drug converting enzymes and/or suicide gene products.
Likewise, Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89:1847-1851 (Lee et al.) and Jones et al., 1992, Infect. Immun. 60:2475-2480 (Jones et al.) isolated mutants of Salmonella typhimurium that were able to invade HEp-2 (human epidermoid carcinoma) cells in vitro in significantly greater numbers than the wild type strain. The xe2x80x9chyperinvasivexe2x80x9d mutants were isolated under conditions of aerobic growth of the bacteria that normally repress the ability of wild type strains to invade HEp-2 animal cells. However, Lee et al. and Jones et al. did not suggest the use of such mutants as therapeutic vectors, nor did they suggest the isolation of tumor-specific bacteria by selecting for mutants that show infection preference for melanoma or other cancers over normal cells of the body. Without tumor-specificity or other forms of attenuation, such hyperinvasive Salmonella typhimurium as described by Lee et al. and Jones et al. would likely be pan-invasive, causing wide-spread infection in the cancer patient. Further, without selection for tumor specificity or employment of other forms of attenuation, use of such bacteria as therapeutic vectors would increase the risk of pan-infection and septic shock to the cancer patient.
Pan et al., 1995, Nature Medicine 1:471-477 (Pan et al.) described the use of Listeria monocytogenes as a vaccine for the immunization of mice against lethal challenges with tumor cells expressing the same antigen expressed by the Listeria vaccine. In addition, they showed regression of established tumors when immunized after tumor development in an antigen specific T-cell-dependent manner. However, Pan et al. did not show that Listeria monocytogenes could be used as a tumor specific vector, which would target and amplify within the tumor. Rather, Pan et al. showed that recombinant Listeria monocytogenes has the ability to deliver a foreign antigen to the immune system and to involve cell-mediated immunity against the same antigen.
Sizemore et al., 1995, Science 270:299-302 (Sizemore et al.) described the use of attenuated Shigella bacteria as a DNA delivery vehicle for DNA-mediated immunization. Sizemore et al. showed that an attenuated strain of Shigella invaded mammalian cells in culture and delivered DNA plasmids containing foreign genes to the cytoplasm of the cells. Foreign protein was produced in the mammalian cells as a result of the procedure. The Shigella vector was designed to deliver DNA to colonic mucosa, providing a potential oral and mucosal DNA immunization procedure as well as other gene immunotherapy strategies. However, Sizemore et al. did not suggest the use of such attenuated Shigella as tumor vectors in that they could be used to target tumors and thereby express genes within them. Rather, Sizemore et al. envisioned its use in vaccination therapy following oral delivery and invasion of the mucosa.
Clostridium was previously investigated as a potential therapeutic vector for solid tumors. The propensity of spores of the obligate anaerobe Clostridium to germinate in necrotic tissues is well known. Tetanus and gas gangrene result from successful colonization of necrotic tissue by pathogenic members of this genus.
Parker et al., 1947, Proc. Soc. Exp. Biol. Med. pp. 461-467 first showed that direct injection of spores of Clostridium histolyticus into a transplantable sarcoma growing in a mouse caused oncolysis, i.e., liquification, as well as regression of the tumor. In general the process of Clostridium-mediated oncolysis was accompanied by acute toxicity and death of the mice. Malmgren and Flanigan, 1955, Cancer Res. 15:473 demonstrated that mice bearing mammary carcinomas, hepatomas, and other tumors died within 48 hrs of intravenous injection of Clostridium tetani spores, whereas control, non-tumor bearing animals were asymptomatic for 40 days. Mxc3x6se and Mxc3x6se, 1964, Cancer Res. 24:212-216 (Mxc3x6se and Mxc3x6se) described the colonization and oncolysis of tumors by Clostridium butyricum, strain M-55, a non-pathogenic soil isolate. Mxc3x6se and Mxc3x6se established the lack of human pathogenicity of the M-55 strain by administering spores to themselves, as reported by Carey et al., 1967, Eur. J. Cancer 3:37-46. Using Clostridium butyricum strain M-55, Mxc3x6se and Mxc3x6se reported that intravenous injections of spores caused oncolysis of the mouse Erlich ascites tumor, growing experimentally as a solid tumor. Aerobic spore-forming organismsxe2x80x94e.g., Bacillus mesentericus, Bacillus subtilis, which were prepared in a similar manner, did not show any oncolysis under the same conditions. Mxc3x6se and Mxc3x6se concluded that the clostridial oncolysis was restricted to anaerobic areas of the tumors because of the anaerobic metabolic requirements of the bacteria.
Gericke and Engelbart, 1964, Cancer Res. 24:217-221 showed that intravenously injected spores of strain M-55 produced extensive lysis of a number of different tumors, but with shortened survival times of the Clostridium-treated, tumor-bearing animals compared to non-treated tumor-bearing animals. Further, they found that xe2x80x9cmetastases in organs or lymph nodes were unaffected by the spores unless the metastatic tumors had reached a considerable size.xe2x80x9d
Thiele et al., 1964, Cancer Res. 24:222-233 showed that intravenously injected spores of a number of species of nonpathogenic Clostridia, including M-55, localized and germinated in tumor tissue, but not in normal tissues of the mouse. Thiel et al., 1964, Cancer Res. 24:234-238 found that spore treatment produced no effect when administered early in the development of the tumor, i.e., when the tumors were of small size. While the spores caused oncolysis in tumors of sufficient size, there was no effect in smaller tumors or metastases. The animals regularly died during oncolysis. Carey et al., 1967, Eur. J. Cancer 3:37-46, concluded that small tumors and metastases had been noted to be resistant to oncolysis whereas large neoplasms were particularly favorable. Thus, the qualitative differences in germination of spores were likely to be not a characteristic of neoplastic and normal tissues per se, but related to physiologic and biochemical conditions found within large tumor masses.
Recent molecular genetic studies have focused on anaerobic bacteria of the genus Clostridium as potential tumor vectors. Fox et al., 1996, Gene Therapy 3:173-178 using a Clostridium expression vector were able to transform the E. coli cytosine deaminase gene into Clostridium beijerincki, which resulted in increased cytosine deaminase activity in the growth medium supernatant and cell extracts of transformed clostridial bacteria. Such supernatants, when added to cultures of mouse EMT6 carcinoma made the cells sensitive to 5-fluorocytosine, presumably through its conversion to the toxic 5-fluorouracil. Similarly, Minton et al., 1996, FEMS Microbiol. Rev. 17:357-364 inserted the E. coli nitroreductase gene into Clostridium beijerincki and were able to detect expression of the gene in an in vivo murine tumor model through the use of antibodies directed against the E. coli nitroreductase gene. The nitroreductase gene product activates CB1954, a potent alkylating agent.
Nothing in any of the above references (or any other references known to the present inventors) suggests the use of any microorganisms, other than the obligate anaerobe Clostridium, as a potential therapeutic vector for solid tumors.
Bacon et al., 1950, Br. J. Exp. Path. 31:703-713; Br. J. Exp. Path. 31:714-724; 1951, Br. J. Exp. Path. 32:85-96 demonstrated that attenuation of Salmonella for virulence in mice can be achieved through auxotrophic mutations, i.e., through the use of mutants which lack the ability to synthesize precursor molecules necessary for growth. More specifically, the authors showed that purine-requiring (Purxe2x88x92) auxotrophs of Salmonella were attenuated in mice.
Hoiseth and Stocker, 1981, Nature 291: 238-239 showed that Salmonella typhimurium auxotrophic mutants with requirements for aromatic amino acids (Aroxe2x88x92) were attenuated for virulence in C57BL mice. Further, Su et al., 1992, Microbiol. Pathogenesis 13:465-476 showed that one such Aroxe2x88x92 mutant, the attenuated antigen carrier strain of Salmonella typhimurium, SL3261, was useful as a vaccine. The Shiga toxin B-subunit/hemolysin A (C-terminus) fusion protein was expressed and underwent extracellular export resulting in antigen-specific immune responses in mice inoculated with these bacteria.
O""Callaghan et al., 1988, Infect. Immun. 56:419-423 characterized Salmonella typhimurium that were both Aro- and Pur- and found that although they were highly attenuated in BALB/c mice, they persisted for several weeks in the livers and spleens following i.v. injections. They were found to be ineffective as vaccines when administered either orally or i.v.
Johnson et al., 1991, Mol. Microbiol. 5:401-407 (Johnson et al.) demonstrated that attenuation in Salmonella virulence can be achieved through mutations in the heat shock inducible protein HtrA, a serine protease. Chabalgoity et al., 1996, Mol. Microbiol. 19:791-801, demonstrated that such attenuated htrA- Salmonella typhimurium were useful as live vaccines.
However, none of the references by Bacon et al., Hoiseth and Stocker, O""Callaghan et al., Johnson et al., Su et al. 1992, Chabalgoity et al. 1996, nor any of the studies referred to in Table 4, infra, suggest that such avirulent strains of Salmonella typhimurium would survive and proliferate within solid tumors, nor that such avirulent mutants might be used as vectors for solid tumor therapy.
The problems associated with the many physical barriers for delivery of therapeutic agents to solid tumors provide clear and difficult obstacles in the design of effective delivery systems. Thus, there has been a long felt need in the art to provide delivery systems which are able to overcome these obstacles.
It is an object of the present invention to use and to provide more advanced biological vectors such as parasites having several distinct advantages as a novel delivery system, some of which are listed below, as well as to meet the challenges of tumor therapy.
Antibiotic Sensitivity: It is an advantage for a tumor-specific parasitic vector to be sensitive to exogenously administered antibiotics. Parasites, such as bacteria, can be eradicated within their hosts by the administration of antibiotics. Such antibiotic sensitivity allows for the eradication of the parasite from the cancer patient""s body upon completion of the therapeutic protocol.
Biospecificity: It is an advantage for a vector to express specificity for its target cell, e.g., a tumor cell. The more specificity, of the vector for the tumor cell, the lower the inoculum necessary for effective therapy, thereby reducing the risk of septic shock or pan-infection to the cancer patient. Parasites show a great degree of natural biospecificity, having evolved to utilize a variety of specific recognition and invasion mechanisms. (For general discussions on biospecificity see: Falkow, 1991, Cell 65:1099-1102; Tumomanen, 1993, Am. Soc. Microbiol. 59:292-296).
Mutant Isolation and Genetic Manipulation: It is an advantage, in the design and isolation of a parasite as a tumor-specific, therapeutic vector, for the parasite to be amenable to genetic manipulation. Parasites with haploid genomes and short generation times, for example, bacteria such as Salmonella typhimurium and enteroinvasive Escherichia coli, can be readily subjected to mutagenesis followed by enrichment procedures for the isolation of strains with desired new characteristics (see generally, Neidhardt et al., (ed.) 1987, Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology. American Society for Microbiology, pp 990-1033. Furthermore, the methods for the genetic analysis and stable introduction of genetic constructs into these bacteria are well known to the science of molecular genetics.
Chemotaxis: A chemotactic response toward cancer cells is an advantage for a tumor-specific vector, for example as a stimulus for the vector to invade through a basement membrane matrix such as that produced by endothelial cells in the vasculature, or as a stimulus for the vector to seek out cancer cells surrounded by tumor matrix. Chemotactic responses in parasites and commensalists or mutualists, particularly in bacteria such as Escherichia coli and Salmonella typhimurium, are well documented. For a review of chemotaxis see Macnab, 1992, Ann. Rev. Genet. 26:131-158.
Replication Within Target Cells: The ability to replicate within target cells is an advantage for a tumor-specific vector. Such an ability allows for amplification of the therapeutic vector number within the infected cancer cell, thus increasing the therapeutic effectiveness of the vector. Progeny of vectors within cancer cells further infect surrounding or distant cancer cells, thus amplifying the vector number within the tumor cell population.
Anaerobic and Aerobic Metabolism: The ability to express invasive and amplification capacities under either aerobic or anaerobic conditions is an advantage for a tumor-specific vector. Solid tumors generally contain vascularized, oxygen-rich areas as well as necrotic oxygen-poor areas. A vector that is functional in both such environments would be able to reach a larger portion of tumor cells than one that can function in only one environment, such as, for example, an obligate anaerobe or aerobe.
The present invention provides compositions and methods for delivery of genes and/or gene products to and/or into target mammalian cells in vitro or in vivo. The genes and/or gene products are delivered by microorganism vectors, including bacteria, fungal and protozoan parasites, which are selected and/or genetically engineered to be specific to a particular type of target mammalian cell. In a preferred embodiment, the vectors function under both aerobic and anaerobic conditions, are super-infective, tumor-specific microorganisms useful for diagnosis or treatment of sarcomas, carcinomas, lymphomas or other solid tumor cancers, such as germ line tumors and tumors of the central nervous system, including, but not limited to, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
Vectors useful for the methods of the present invention include but are not limited to Borrelia burgdorferi, Brucella melitensis, Escherichia coli, enteroinvasive Escherichia coli, Legionella pneumophila, Salmonella typhi, Salmonella typhimurium, Shigella spp., Streptococcus spp., Treponema pallidum, Yersinia enterocohtica, Chlamydia trachomatis, Listeria monocytogenies, Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis, BCG, Mycoplasma hominis, Rickettsiae quintana, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis carnii, Eimeria acervulina, Neospora caninum, Plasmodium falciparum, Sarcocystis suihominis, Toxoplasma gondii, Leishmania amazonensis, Leishmania major, Leishmania mexacana, Leptomonas karyophilus, Phytomonas spp., Trypanasoma cruzi, Encephahtozoon cuniculi, Nosema helminthorum, Unikaryon legeri. 
As used herein, Salmonella typhimurium encompasses all Salmonella species. It has long been recognized that the various xe2x80x9cspeciesxe2x80x9d of the genus Salmonella are in fact a single species by all acceptable criteria of bacterial taxonomy. The single species is now designated xe2x80x9cSalmonella entericaxe2x80x9d. F. Neidhardt (ed.), Escherichia coli and Salmonella, 1996, Volume I, pp. xx, ASM Press, Wash. D.C.
An embodiment of the present invention is to provide methods for the isolation of super-infective, attenuated, tumor-specific mutants of microorganisms such as bacterial, fungal and protozoan parasites. Further, the present invention provides methods for use of these microorganisms in the diagnosis and treatment of malignant and/or metastatic solid tumor cancers, such as melanoma or colon cancer. Moreover, these mutant parasites may express specific gene products, some of which are secreted into the cytoplasm or vacuolar space of the infected cell.
The present invention provides methods for the isolation of super-infective target cell-specific microorganisms. In particular embodiments, the invention provides for the isolation and use of super-infective, tumor-specific strains of parasites such as the bacterium Salmonella spp., including S. typhimurium, the bacterium Mycobacterium avium, and the protozoan Leishmania amazonensis. The tumor-specific vectors can also contain suicide genes.
One embodiment of the present invention provides methods for the isolation of and compositions comprising super-infective, tumor-specific mutants of Salmonella spp., e.g., Salmonella typhimurium, and for their use in the diagnosis and treatment of sarcomas, carcinomas, melanomas, colon cancer, and other solid tumor cancers. Another embodiment of the present invention provides methods for the isolation of and compositions comprising super-infective, tumor-specific mutants of Salmonella spp. containing a suicide gene. In a specific embodiment, the suicide gene is thymidine kinase from Herpes simplex virus or cytosine deaminase from Escherichia coli or human microsomal p450 oxidoreductase.
Another embodiment of the present invention provides methods for the isolation of and compositions comprising super-infective, tumor-specific mutants of the protozoan, Leishmania amazonensis and for their use in the diagnosis and treatment of sarcomas, carcinomas, melanomas, colon cancer, and other solid tumor cancers.
Yet another embodiment of the present invention provides methods for the isolation of and compositions comprising super-infective, tumor-specific mutants of the bacterium Mycobacterium avium and for their use in the diagnosis and treatment of sarcomas, carcinomas, melanomas, colon cancer, and other solid tumor cancers.
Yet another embodiment of the present invention provides methods for attenuation of parasite vector toxicity so as to reduce the risk of septic shock or other complications in the host, i.e., the patient receiving vector-delivered gene therapy. Such methods include mutagenesis of parasites; isolation of parasite mutants with increased tumor specificity, increased specificity for suicide gene expression and concomitant reduced ability to infect normal host cells in the body; isolation of mutants with enhanced chemotactic abilities toward cancer cell secretory products; isolation of mutants with genetically altered lipopolysaccharide composition; and isolation of mutants with altered virulence genes so as to achieve specific survival of the parasitic vector in cancer cells as opposed to normal cells of the host body.
The present invention further encompasses use of microorganism vectors for diagnosis or treatment of solid tumor cancers.
The present invention may be understood more fully by reference to the following definitions, detailed description of the invention, illustrative examples of specific embodiments and the appended figures in which:
Attenuation: Attenuation, in addition to its traditional definition in which a microorganism or vector is modified so that the microorganism or vector is less pathogenic, is intended to include also the modification of a microorganism or vector so that a lower titer of that microorganism or vector can be administered to a patient and still achieve comparable results as if one had administered a higher titer of the parental microorganism or vector. The end result of attenuation is that the risk of toxicity as well as other side-effects is decreased, when the microorganism or vector is administered to the patient.
Suicide gene: A suicide gene is defined as a gene that when delivered to a target cell and expressed by a vector of the present invention causes the death of the target cell and/or the vector.
Super-infective: A super-infective vector is defined as a vector which is able to attach and/or infect a target cell more readily as compared to the wild type vector. Depending on the population density of the inoculum, the ratio between super-infective vectors and wild type vectors detectably infecting a target cell approaches 4:1, preferably 30:1, more preferably 90:1. Most preferably, one is able to reduce the inoculum size and infection time so that only the super-infective vectors have time to attach to and/or infect cancer cells growing in cell culture in vitro or as tumors in vivo.
Tumor-specific: A tumor-specific vector is defined as a vector which is able to distinguish between a cancerous target cell and the non-cancerous counterpart cell so that the vector preferentially attaches to, infects and/or remains viable in the cancerous target cell.